Osteoclasts differentiate from recirculating monocytes in niches adjacent to bone and require two critical signals, M-CSF and RANKL. How monocytes and osteoclast precursors migrate to the bone to interact with cells expressing the appropriate signals is unknown. Upon receiving these signals, osteoclast precursors undergo a differentiation process that requires cell-cell interaction and multiple cell fusion events. As cel-cell interaction and fusion is a dynamic process it predicts that osteoclast precursors continue to be motile after receiving differentiation signals. However, the mechanism(s) regulating monocyte/osteoclast precursor motility and migration to bone surfaces remain unresolved. Cell motility is largely controlled by Gi-protein coupled receptors (GiPCR). We have recently uncovered that osteoclast precursors and osteoclasts robustly express EBI2 (Gpr183, a GiPCR) and EBI2 ligand is produced by cells located within osteoclastogenic bone marrow niches. Interestingly, osteoclasts derived from EBI2 -deficient mice in vitro are defective in size and EBI2 deficiency results in increased bone density and protects mice from ovariectomy- induced osteoporosis. Previous studies demonstrated that EBI2 and its ligands direct B cell migration to outer and interfollicular niches in secondary lymphoid organs during primary T-dependent antibody response. Therefore, we considered the possibility that EBI2 promotes BMDM and osteoclast precursor motility thereby increasing the probability of cell-cell interaction and cell-cell fusion essential for differentiation of large multinucleated osteoclasts. In preliminry experiments we found that EBI2-deficient cells moved with slower velocity and exhibited reduced displacement over time in vitro. Finally, our preliminary studies show that osteoblasts secrete substantial amounts of EBI2-ligand, which may promote directional migration of EBI2-expressing osteoclast precursors to bone niches. Indeed, retroviral overexpression of EBI2 in osteoclast precursors preferentially positions these cells in closer proximity to the bone surface than in control cells. A major focus of this proposal is to uncover the molecular mechanisms underlying EBI2 regulation of monocyte/osteoclast precursor motility and migration to sites of osteoclastogenesis. We will use a combination of 2-photon and cellular imaging approaches to characterize the role played specifically by EBI2 (Gpr183, a GiPCR) in osteoclast differentiation, motility, and migration to the bone surface. Given that GPCRs are prime targets for therapeutic interventions, the proposed studies have the potential to elucidate new aspects of osteoclast biology that should have a translational impact in bone disease.